Antibiotic xk&#39; 33&#39; f&#39; 2 &#39;and process for producing same

ABSTRACT

A new antibiotic designated XK-33-F2 is produced by culturing strains of microorganisms belonging to the genus Streptomyces, which are capable of producing the antibiotic, in a nutrient medium. The antibiotic is thereafter separated and recovered from the medium.

United States Patent 11 1 Nara et al.

Aw 3,901,972 Aug. 26, 1975 ANTIBIOTIC XK-33 F AND PROCESS FOR PRODUCING SAME [75] Inventors: Takashi Nara, Tokyo; Seigo Takasawa, Kawasaki; Ryo Okachi, Machida; Isao Kawamoto, Machida; Masaru Kumakawa, Machida; Mitsuyoshi Yamamoto, Machida; Seiji Sato, Machida, all of Japan [73] Assignee: Abbott Laboratories, North Chicago, Ill.

22 Filed: Dec. 26, 1972 211 Appl. No.: 318.337

Related US. Application Data [63] Continuation-impart of Ser. No. 212,620, Dec. 27,

197 l abandoned,

[52] U.S. Cl 424/ll6; 195/80 [5]] Int. Cl Afilk 2l/00 [58] Field of Search 424/1 [6; l95/80 [56] References Cited 7 OTHER PUBLICATIONS Miller, Pfizer Handbook of Microbial Metabolities, McGraw-Hill Book Co., Inc, N.Y., N.Y., l96l, p. 45l.

Primary Examiner-Jerome D. Goldberg Attorney, Agent, or Firm-Gildo E. Fato; Robert L. Niblack [57] ABSTRACT A new antibiotic designated XK-33-F is produced by culturing strains of microorganisms belonging to the genus Slreptomyces, which are capable of producing the antibiotic, in a nutrient medium. The antibiotic is thereafter separated and recovered from the medium.

2 Claims, 2 Drawing Figures ANTIBIOTIC XK-33-F AND PROCESS FOR PRODUCING SAME CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of copending application U.S. Ser. No. 2l2,620. filed Dec. 27, 1971, now abandoned.

BACKGROUND OF THE INVENTION The present invention relates to a process for producing a new antibiotic XK-33-F by fermentation and more particularly. to a process which comprises culturing a strain of microorganism belonging to the genus Srreptomyces, which is capable of producing the antibiotic XK-3 3-F in a suitable medium to form said antibiotic, and recovering same from the culture liquor.

inventors have developed a process for production of this antibiotic.

SUMMARY OF THE INVENTION In accordance with the present invention, a new antibiotic designated XK33F is produced. The antibiotic is produced by strains of microorganisms belonging to the genus Srreptomyces. A particularly suitable microorganism is slrcptumyc'es olimrericuli var. celluloplu'lus which has been newly found and named by the present inventiors. its typical strain, which has been isolated from soil found in a district in Japan. has been deposited with the American Type Culture Collection in Roekville. Md. and has been accorded accession number 2l632.

The above-mentioned strain is characterized morphologically by growth which is yellowish brown with grayish white or slightly greenish cream-colored aerial mycelia. Formation of melanine is observed on various New antibiotics are always in demand and particu- 20 media. Sporophores are secondarily formed from the larly those which are effective against a broad spectrum of bacteria are greatly sought after. However, even when a new antibiotic is found, it must be producible on an industrial scale in order to be economically feasible. To this end. the present inventors have isolated a 25 aerial myeelia and they form whorls. In most cases the spores form in line by ten or more. forming a flexuous curve; and spore surface is smooth. No spirals are observed.

Culturing characteristics of the above-noted strain after culturing respectively on IS kinds of media at 36C for 2 weeks are shown in the following TABLE l.

TABLE 1 i Name of Growth Substrate Aerial Formation Soluble medium mycclium myeelium of aerial pigments myeelium Yeast extract good brown gray moderate none malt extract S: 3 h (in agar R:( 3 hg) Starch agar good brown gray moderate none S:( 3 ng) (d1 R:(3 ngl Glycerol good yellowish greenish moderate none asparaginc brown creamagar S:( 2 le] colored R:(Zle) lzhill C mpek s agar poor colorless gray poor none Glucose moderate brown creampoor brown asparagine S:( 2 le) colored (3 ne) agar R:( ng) (2 ca) Nutrient agar good brown gray moderate hrown S:(] nil (2th) (3 ni) R:( 3 ni) Glycerol good thin greenish poor none calcium yellowish creammalate agar green colored S:( 1 ca) I ha) R:(l ea) Plain agar poor colorless white poor none TABLE l-Continued Name of Growth Substrate Aerial Formation Soluble medium myeelium myeelium of aerial pigments mycelium Egg albumin poor colorless colorpoor none agar less Tyrosine agar moderate brown creampoor none S13 le) colored R:(3le] (Zeal Peptone good greenish greenish moderate greenish glucose agar brown gray brown SzHV. (ldc) (Zli) lg) R:(2 Ii) Gelatin moderate greenish greenish moderate brown brown cream- Sz 2 nil colored R:(2ni) (l ha) Milk moderate brown gray poor Reddish Sit. g) (b) pigment R13 lg) was ol'vservcd Glucose moderate Grow in greenish poor none Czapek the liquor creamliquor as small colored greenish I ha) creamcolored spheres Filter moderate colorless none none paper cellulose Potato good gray greenish moderate brown S:( 2 cream- (4 li) R13 l1) colored I 2 ha) Cellulose moderate greenish moderate none I powder I brown meat peptone 2 lg] agar R12 lg) Loeftlers good brown none hlakish serum S12 ie) brown R15 pm (4 at) only slightly In the above TABLE: The indication in the parentheses are in accordance with the color classification based on C lor Harmon Manual (Container Comm 1 of America )1 and '8: color of the surface of the substrate nneelium R1 color of the ruerse side oithe suhstrate m celinm The assimilability of carbon sources of the present strain is shown in the following TABLE 2.

TABLE 2 ASSIMILABILITY OF CARBON SOURCE C arhon Assimilahility Carbon Assimilahility source source Glucose -H Raftinose Fructose Mannitol Lactose Rhamnose i Arahinose Sucrose lnositol -H- Mannosc +4- Glyeerol -H- Saliein Xylose (all 8. Reduction of nitrates to nitrites: negative 9. Decomposition of cellulose: strongly positive The properties of the present strain were compared with those of the known Actinomycetes described in "The Actinomycetes." Vol. ll. by Waksman'. and the 5 present strain has been determined to belong to "series reticuli." Of the known species in this series, the following three species are referred to as those most closely related.

I. .S'rrepmnn'ces olivm'eticuli l 2. Slrepmmyt'es rerricillarus 3. .Srrepmnrvces [ureuvertz'cillulux A comparison of the properties of the present strain and those ofthe closely related species are shown in the Culturing is carried out in a liquid culture medium and the submerged-stirring culture method is particularly suitable. Generally. the ordinary methods for culturing microorganisms of the Actinumycetes may be used. As for the culture medium either natural or synthetic medium may be used so long as it contains a carbon source, a nitrogen source, inorganic compounds and small amounts of additional nutrients necessary for the specific microorganism. As a carbon source. glucose. starch. glycerol, mannose, fructose. inositol. mannitol. sucrose. molasses, for example. may be used alone or in combination. In addition. hydrocarbons, alcohols, organic acids, etc. are also appropriate depending upon the assimilability thereof by the specific mifollowing TABLE 3. croorganism. As organic and inorganic nitrogen TABLE 3 Nutrient gray grayish white no yellowish white agar acrial mycclium aerial mycclium aerial mycclium aerial mycelium Potato greenish creamgreenish creamyellowish green colored colored aerial mycclium aerial mycclium aerial mycclium Litmus milk gray aerial coagulatcd. pepcoagulated. brown aerial mycclium. coagu tonized. pH; unpcptonized mycelium latcd. peptonizchanged Coagulation is uned. alkaline pH certain but pcptonization is clear.

Cellulose decomposed not decomposed not decomposed Reduction of negative negative positive nitrates Assimilation mannitol (-1 mannitol mannitol of carbon sources lactose lactose (+1 lactose Productive XK-33F.

capacity of viomycin.

antibiotics an antibiotic similar to viom cin destomycin A Since the microorganism of the present invention appears to have a closer resemblance to .S'trepmnqvcm olimreticuli rather than to Slrepmmyt'es verticillams or .S'rreptonrvz'es [ureurerricillatus and since it resembles Srrepmmyces ulimrericuli in most characteristics except those noted above. it has been identified as a variety of Strepmnrvt'es olii'm'etic-uli and named Strepmmyces ulii'urericuli var. celluluphilus.

As is the case with other strains of Acrinomyces. the strains of the present invention can be mutated by artificial means such as ultraviolet ray irradiation. Co ir radiation. X-ray irradiation and treatment with various mutation inducers. Accordingly. any strain belonging to Slrepmnrvces olivorericuli var. cellulop/iilus and Capable of producing XK33-F including mutants thereof can be used in the present invention.

60 phosphates. etc. may be added to the medium and other organic or inorganic substances capable of promoting growth of the specific microorganism may also be added to the medium.

Culturing is carried out at a temperature of between 65 to 40C. and a temperature within the range of to C. is generally preferred. in addition. the pH should be maintained approximately neutral for best results.

It is preferred that the microorganism be initially grown in a seed medium prior to being used for inoculation of the culture medium. The seed medium is incubated under favorable growth conditions for a period of time sufficient to develop a suitable organism population. The seed medium is then used to inoculate the culture medium.

Culturing is carried out usually for 2 to 7 days and the present antibiotic is formed and accumulated in the culture liquor. When the amount of antibiotic formed in the culture liquor reaches the maximum. culturing is discontinued, and the desired product is isolated from the culture liquor after the microbial cells are removed therefrom such as by filtration.

A suitable method for isolating the antibiotic from the culture filtrate comprises a chromatography using an ion exchange resin column. For example, the culture filtrate, obtained by filtering the culture liquir together with a filter aid of the Celite type, is adjusted to pH 8, and adsorbed on a weakly acidic cation exchange resin such as Amberlite [RC-50 (NH, form). After washing with water. elution is initially carried out with 0.5 N NH,OH, whereby only XK-33-F, is eluted. XK-Ii3--F is an antibiotic similar to destomycin A. Thereafter, elution is carried out with 0.5 N HCl, whereby two fractions are separately obtained. The first elution fraction containing our antibiotic XK33F and the second elution fraction contains an antibiotic XK-33-F;,. which is identified to be viomycin.

The fraction containing XK33F is adjusted to pH 8.0 and then is subjected to a column chromatography with carbon powders. After elution is carried out with 0.5 N HCl 80 72 MeOH, the resulting cluate is passed through a column of an anion exchange resin such as Dowex l X 2 (OH form) and eluted with water. By freeze-drying an active fraction of the eluate, a white free base of XK-33-F is obtained. The thus obtained substance is relatively stable in a neutral state as well as in an acidic state, but slightly unstable in an alkaline state.

The novel antibiotic produced in accordance with the present invention is hereafter further described with reference to the accompanying drawings wherein:

FIG. 1 illustrates the ultraviolet absorption spectrum of the antibiotic of the present invention; and

FIG. 2 illustrates the infra-red absorption spectrum of the antibiotic.

The antibiotic produced in accordance with the present invention is a white basic powder which does not exhibit a distinct melting point, but turns brown at 200C and decomposes at 205 210C. [t is soluble in water and slightly soluble in methanol, but insoluble in such organic solvents as ethanol, butanol, acetone, benzene. ethyl acetate, chloroform, ether, petroleum ether, hexane, etc.

Referring to P16. 1, the ultraviolet absorption spectrum of the antibiotic shows a maximum absorption at 268 my in an aqueous solution and N/lO HCl, and at 283 my in N/lO NaOH. The elementary analysis of hydrochloride of the present substance reveals C; 33.28 72, H; 5.51 7r and N: 2|.73 7r. Further, its specific rotation [01],, is -l2.4 (C=l, H O).

The present antibiotic is positive in ninhydrin reaction and Sakaguchi reaction and exhibits yellow color with potassium permanganate, but is negative in Fehling reaction. Molisch reaction and anthrone reaction.

The infra-red absorption spectrum of the present substance is illustrated in FIG. 2. It shows characteristic peaks at the following wave lengths expressed in reciprocal centimeters: 945, 985, L050, 1,160, 1,230, 1,330, 1,500, 1,670 and 3,300.

As a further identification, the Rf values obtained when the present substance is subjected to a thin layer chromatography (TLC) of silica gel using three kinds of developers are given in the following TABLE 4.

( l is upper layer portion of a mixture of chloroform, methanol and l7 1 aqueous ammonia (2:1:l 1

[2) mixture of H) '55 ammonium acetate and methanol l:l

l 3) is a mixture of It) ,6 ammonium acetate. acetone and Ill '1 aqueous ammonia lfihlllzllsl The antimicrobial spectra of the present antibiotic to various microorganisms are given in the following TABLE 5.

TABLE 5 M icroo rgu n ism s tested Minimum inhibiting concentration by agar dilution method y/ l 1 Minimum inhibiting concentrationt 'ylml TABLE -Continued Minimum inhibiting concentration by agar dilution method (v/mll Minimum inhibiting Microorganisms tested concentration 14ml) As shown above, the present antibiotic is effective upon such Gram-positive bacteria as Bacillus subtilis, Bacillus cereus and the like, such Gram-negative bacteriaas Klebsiella pneumoniae, Escherichia coli, Salmonella typhosu. etc., or acid-fast bacteria such as Mycobacren'um pheli, etc. However, it is ineffective upon molds, yeasts, etc.

Moreover, in a bioassay in vitro, it is also effective upon Mycoplasma gallisepricum which is resistant to spiramycin.

The present antibiotic was also tested in vivo. Specifically, mice were infected with E. coli. A group of ten mice were administered subcutaneously at l and 6 hours with a total of 1,200 mgJkg. of the antibiotic of the instant invention. All l0 untreated mice died after 24 hours. Eight of the It) mice treated with the antibiotic here survived after the 24 hour period.

As compared with the well-known antibiotics, the present substance XK-3 3-H. is similar to antibiotics of the group of viomycin. tuberactinomycin and tuberactinomycin N in the patterns of ultraviolet absorption spectrum, elementary analysis, solubility, infra-red absorption spectrum and antimicrobial spectra. A more 40 detailed comparison is given in the following TABLE TABLE 6 Rf value of thin layer chromatography on silica gel XK-SI -F- Viomycin Tuberactino Tuheructinm mycin myein N Developer l) 0.50 0.12 0. I2 0. l 2 Developer (2] 0.67 (1.67 0.67 0.67 Developer (3] 0.7l (1.36 0.58 0.58 Ultraviolet absorption MaX(E 268" (348) 2b8(339) 268(3 l0) 268(342) (in H O, (in N/ltl (in H. .O) (in M 0, N/IU HCI) HCI] N/ltl HCI) 283" (237) ISO-2M6 268.5(250) 288(215) (in N/lU (219) (in HCl) (in N/IU NaOl-l) (in N/lU 285.5(180) NaOH) NaOH) (in NaOH) Elementary analysis C ('1) 312K 4|.75-418l 35.64 37.70 H 5.5] 6.35- 6.99 5.96 6.12 N 21.73 25.78-24.96 l7. [4 22.50

(hydrochloride. (sulfate) (Q2133!) ash 0.14 '1 l [11],, l2.4 33.5 3l.5 l'-).l

(C=l H 0 (h ydrohydrohydro- (hydro chloride chloride] chloride) chloride I (olor reaction Ninhydrin H- (+1 Sahaguchi H) (-l NMnO, (+1 H) (H Toxicity LO (mice. intraveneous) 741] rug/kg 240 mg/kg Hi3 mg/kg 385 mg/kg References:

'IJ. Friedman: U. S. Patent 23127.? W58) A. Nuguul el illi J. Antibiotics 2|. Nil 4 Whit) "laku i Amlo et ul; IIh meeting of antibiotic-stud (September 25. I97") As is clear from the data of TABLE 6. the Rl' values of silica gel thin layer chromatography with the developers l) and (3) shown in TABLE 4 are oboviously different from those of viomycin. tuberactinomycin and tuberactinomycin N. Further. [aj of XK--33-F- is lowest and is different from those of the other three. ln addition. the elementary analysis of the antibiotic of the present invention indicates a carbon content which is lower than known antibiotics. Furthermore. the antimicrobial activity of the present substance is lower than those of said three antibiotics and is about a few fractions or about one-tenth of those of the latter. On the other hand. the toxicity ofthe present substance is considerably lower than those of said other three.

Practice of a specific embodiment of the present invention is illustrated by the following representative example.

EXAMPLE 1 In this example. a strain ATCC 21632 of Streptomycex olirureliculi var. celluluphilus is initially cultured in a seed medium containing 2 "/1 glucose, 0.1 yeast extract. 0.5 7: peptone and 0.] 7r CaCQ, (pH 7.2 before sterilization). One loop of the seed microorganism is inoculated into ml of the said seed culture medium in a 250 ml Erlenmyer flask and culturing is carried out with shaking at 30C for 48 hours. 30 ml of the thus obtained seed culture liquor is inoculated into 300 ml of a second seed culture medium comprising 2 7c glucose. 2 7r defatted soybean powder and ().l 7! CaCO,-, (pH 7.2 before sterilization), in a 2 l Erlenmyer flask provided with baffles.

The second seed culture medium is cultured with shaking at 30C for 48 hours, Then, 900 ml of second seed culture liquor is inoculated into I7 I of the main fermentation medium having the same composition as the second seed medium, in a 30 l glass jar fermenter. The jar fermenter culturing is carried out at 30C for 72 hours by the aeration-stirring method (300 rpm; aeration rate: l5 l/min).

After 72 hours of culturing the antibiotics. XK-3- 3-F Xl'(-33--F (a substance similar to destomycin A) and XK-33-F (viomycin) are formed in the culture liquor.

To isolate the antibiotics, 16 l of the culture liquor is added to L5 kg of Radiolite 600 (produced by Showa Chemical Industry CO., Ltd. a filter aid ofCelite type. and stirred for 15 minutes and filtered. Then, 1 l l of the culture liquor (filtrate) is adjusted to pH 8 and passed through a column of 500 ml of Amberlite lRC 5()(NH form). After washing with about 3 l of water. elution is carried out initially with 0.5 N NH OH and about 3 l of eluate is obtained. This fraction contains the substance XK-33-F which is an antibiotic similar to destomycin A. Then, elution is further carried out with (1.5 N HCl. In the first place, XK33F appears in the elution fraction between I50 ml and 200 ml. and then XK33F,, appears in the elution fraction between 450 ml and 850 ml. XK--33-F is identified to be viomycin. The fraction of XK-33-F; is adjusted to pH 8 and passed through a column of I00 ml of active carbon. The active carbon is one of the types adapted for chromatography which is produced by Wako Pure Chemical Industries Ltd. The adsorbed substance is thereafter eluted with a 0.5 N HCl 76 methanol solution after washing with water (300 ml). The active fraction is passed through a column of 50 ml of Dowex l X 2 (OH form) produced by Dow Chemical Co, for neutralization. By freeze-drying the solution, about l g of white XK33F- powder is obtained. The powder is then dissolved in warm methanol and a pure product of XK-3 3F- can be obtained by adding acetone thereto. Physicochemical and biological properties of the present substance are those as already described before.

What is claimed is:

1. An antibiotic XK-33-F produced by the fermentation of Streptomyces ulivoreticuli var. celfulophilus in a nutrient medium. said antibiotic being characterized by an elementary analysis of its hydrochloride of C: 33.28%, H: 5.51%. N: 21.73% and a specific rotation [01],, of l2.4 (C=l, H 0), and having an infra-red absorption spectrum according to FIG. 2 of the drawings.

2. A process for producing the antibiotic designated XK-33-F which comprises: culturing the microorganism Slrepmmyc'cs olirorericuli var. cellulop/tilus at a temperature ranging from about 20 to about 40 C. for 2 7 days in a substantially neutral nutrient medium containing a carbon and nitrogen source; accumulating said antibiotic; and separating and recovering said antibiotic. 

1. AN ANTIBIOTIC XK-33-F2 PRODUCED BY THE FERMENTATION OF STREPTOMYCES OLIVORETICULI VAR. CELLULOPHILIS IN A NUTRIENT MEDIUM, SAID ANTIBIOTIC BEING CHARACTERIZED BY AN ELEMENTARY ANALYSIS OF ITS HYDROCHLORIDE OF C: 33.28%, H: 5.51%, N: 21.73% AND A SPECIFIC ROTATION (A)D25 OF -12.4* (C=1, H2O), AND HAVING AN INFRA-RED ABSORPTION SPECTRUM ACCORDING TO FIG.2 OF THE DRAWINGS.
 2. A process for producing the antibiotic designated XK-33-F2 which comprises: culturing the microorganism Streptomyces olivoreticuli var. cellulophilus at a temperature ranging from about 20* to about 40* C. for 2 - 7 days in a substantially neutral nutrient medium containing a carbon and nitrogen source; accumulating said antibiotic; and separating and recovering said antibiotic. 